Studies inC. eleganshave begun to reveal new components and new
mechanisms associated with intracellular membrane traffic in a variety of cell types.
The worm benefits from many of the advantages of yeast as a genetically tractable
organism for these kinds of studies while offering the unique opportunity to probe how
these pathways have been extended and modified in the context of a multicellular animal
undergoing development to produce diverse cell types such as muscles, nerves, and
polarized epithelia. This review summarizes recent work elucidating endocytic pathways,
primarily in the worm germ line and coelomocytes, and also touches on diverse studies of
secretion, especially in ectodermal cells of epithelial character.

1. The endocytic pathway

Endocytosis is the vesicle-mediated process used by all cells to internalize
extracellular macromolecules, plasma membrane lipids, and plasma membrane proteins (Figure 1). There are several endocytic pathways that utilize different mechanisms to
internalize portions of the plasma membrane. The best studied endocytosis pathway in
worms (Fares and Grant, 2002) and mammalian cells (Brodsky et al., 2001) is the clathrin-coated pit pathway (Figure 2). Many receptors and their
associated ligands cluster into clathrin-coated pits by association with clathrin
adaptor proteins such as the four-subunit complex AP2. Clathrin adaptors in turn bind to
the clathrin lattice which is thought to provide the force required to deform the
membrane into a curved bud. The large GTPase dynamin is then involved in pinching off
the coated pit to form a clathrin-coated vesicle. Such vesicles are then uncoated by the
chaperone hsc70 and the DNA-J domain co-chaperone auxillin. Uncoated endocytic vesicles
then fuse with one another and with early endosomes in a reaction requiring the small
GTPase Rab5. In early endosomes some ligand-receptor complexes dissociate due to the
reduced pH of the endosomal lumen. Many receptors then recycle to the plasma membrane
either directly or indirectly via recycling endosomes. Many ligands do not recycle but
instead are transported from early to late endosomes and eventually to lysosomes for
degradation. Early to late endosome transport may be mediated by small vesicular
intermediates, or may be a maturation process whereby early endosomes lose components
through recycling pathways and gain components through fusion with vesicles derived from
the secretory pathway. Late endosomes are then thought to fuse with pre-lysosomes to
form “hybrid” organelles, which mature back into lysosomes
through sorting and fission.

Figure 1. General model of the endocytic pathway. Cargo molecules are endocytosed and targeted to early endosomes. Some cargos are further transported to lysosomes through
late endosomes. Others are recycled back to the plasma membrane via the recycling pathway.

1.1. Endocytosis in oocytes

One important model for mechanistic studies of endocytosis in C.
elegans focuses on oocytes, which internalize huge quantities of yolk
proteins and their associated lipids by clathrin-mediated endocytosis (Grant and Hirsh, 1999). This process is most easily followed by fluorescence
microscopy using strains that express transgenes encoding the major yolk protein
YP-170 (aka VIT-2) fused to GFP (Figure 3; Grant and Hirsh, 1999).
YP170-GFP is synthesized in the intestine of adult hermaphrodites and is secreted
basolaterally into the body cavity (pseudocoelom). YP170-GFP, like endogenous yolk,
is a cholesterol binding/transport protein related to human ApoB-100, the major
protein component of serum low-density lipoprotein (LDL). The yolk receptor in
C. elegans is RME-2, an LDL-receptor related molecule
expressed specifically in the oocytes (Figure 3; Grant and Hirsh, 1999). RME-2 contains a typical NPXY internalization motif in its intracellular
domain that is known to direct other members of the LDL-receptor family into
clathrin-coated pits. Trafficking of yolk and yolk receptors also depends critically
upon the activities of the endocytic Rab proteins RAB-5, RAB-7, and RAB-11, known
modulators of endocytosis in all eukaryotes (Grant and Hirsh, 1999).
Thus yolk uptake provides a genetically tractable system for the study of clathrin
and Rab dependent transport processes.

Figure 2. Mechanism of clathrin-dependent endocytosis. Clathrin and cargo molecules are assembled into clathrin-coated pits on the plasma membrane together with an adaptor complex
called AP-2 that links clathrin with transmembrane receptors, concluding in the formation of mature clathrin-coated vesicles
(CCVs). CCVs are then actively uncoated and transported to early/sorting endosomes.

Figure 3. YP170::GFP endocytosis by oocytes. YP170::GFP is synthesized in the intestine and secreted into the body cavity from which it is endocytosed by oocytes. RME-2 is the yolk receptor expressed in oocytes. Fluorescent micrographs of wild-type and typical rme mutant worms expressing YP170::GFP are shown.

Screens for mutants defective in YP170-GFP uptake by oocytes identified 11
rme genes (receptor-mediated endocytosis defective) required for various steps in endocytic transport
(Figure 3; Grant and Hirsh, 1999). In contrast to secretory defects,
defects in endocytosis per se do not severely affect the
organization or morphology of the germ line or oocytes as judged by Nomarski optics.
The best studied of the general endocytosis regulators identified in this screen are
RME-1 and RME-8 (Grant and Hirsh, 1999; Zhang et al., 2001). Both of these proteins are found in the cytoplasm of most C.
elegans cells and are associated with the limiting membrane of endosomes. RME-1 is an EH domain protein associated with recycling endosomes and is
thought to contribute to the formation of membrane tubules that recycle receptors
from endosomes to the cell surface (Grant et al., 2001). There are
several mammalian homologues of RME-1, at least one of which functions in recycling
endosomes (Lin et al., 2001). In the absence of RME-1, yolk receptors
become trapped in endosomes of the oocyte and cannot efficiently recycle through
multiple rounds of endocytosis. rme-1 mutants are also
defective in endocytosis by coelomocytes and in basolateral endocytic traffic in the
intestine (Grant et al., 2001).

RME-8 is a large DNA-J domain protein (Zhang et al., 2001). The
precise step in trafficking regulated by RME-8 remains unclear, since the protein
appears to localize to early and/or late endosomes, but rme-8
mutants appear defective in the earliest detectable internalization step (Zhang et al., 2001). Recent studies in Drosophila indicate similar
localization and transport defects in Dm-rme-8 mutants (Chang et al., 2004). These studies also indicated that RME-8 may interact
with Hsc70 and clathrin. J-domains are known to mediate interactions with heat shock
protein 70 family members, and clathrin-coated vesicles are uncoated by the action
of cytoplasmic Hsc70 (Brodsky et al., 2001). Previous work in
C. elegans demonstrated that yolk endocytosis depends upon
the C. elegans auxillin homologue dnj-25,
an other J-domain protein. RNAi of Ce-auxillin results in
abnormal accumulation of clathrin-coated structures and interferes with clathrin
dynamics in vivo (Greener et al., 2001).

1.2. Endocytosis in coelomocytes

The other major model developed for mechanistic studies of endocytosis in
C. elegans focuses on coelomocytes, 6 macrophage-like
scavenger cells in the body cavity (pseudocoelom) that are highly active in
endocytosis of fluid-phase molecules (Fares and Greenwald, 2001).
While the natural molecules that are removed from the body cavity by coelomocytes
are unknown, coelomocytes are capable of removing many foreign compounds from the
body cavity after they are introduced by micro-injection (Fares and Greenwald, 2001). The most common of these endocytosis tracers are fluorescently
labeled BSA or dextrans. These observations allowed the development of a pulse-chase
type assay of coelomocyte endocytosis (Zhang et al., 2001).
Fluorescently labeled BSA or dextran trafficking through the endosomes of
coelomocytes is monitored as a function of time after introduction into the body
cavity. Expression of RME-8:: GFP is often used to facilitate visualization of
endosomes during fluorescent tracer trafficking from the cell surface to the
lysosomes (Zhang et al., 2001).

A simpler and more generally useful assay for coelomocyte endocytic function was
developed by Fares and Greenwald (Figure 4; Fares and Greenwald, 2001a;
Fares and Greenwald, 2001b). They created transgenic worms expressing
signal sequence-GFP fusion protein in body-wall muscles
(pmyo-3::ssGFP). In this strain, GFP is constitutively secreted
from body-wall muscle cells into the pseudocoelom and is then efficiently taken up
and degraded by coelomocytes. At steady-state this strain shows weak pseudocoelom
labeling and stronger labeling of late endosome and lysosome structures within
coelomocytes. Using this assay, cup (coelomocyte uptake defective) mutants were identified that show accumulation of GFP in
the pseudocoelom, and/or aberrant morphology of endocytic organelles within
coelomocytes (Figure 4; Fares and Greenwald, 2001b).

Interestingly, all three cup genes cloned so far encode worm
homologues of human disease genes. CUP-5 is a homologue of human mucolipin-1,
mutations in which lead to Mucolipidosis type IV (Fares and Greenwald, 2001). This family of proteins is thought to act as
Ca2+ channels in endosomal or lysosomal membranes.
cup-5 mutants show severe defects in lysosome biogenesis
that result in the formation of grossly enlarged vacuoles accumulating late
endosome, lysosome, and Golgi markers (Treusch et al., 2004). The
most likely explanation for this phenotype is that CUP-5 is required for
Ca2+ release during the reformation of lysosomes from
late endosome-lysosome hybrid organelles.

Figure 4. GFP endocytosis by coelomocytes. Signal sequence-tagged GFP is synthesized in muscle and secreted into the body cavity. Coelomocytes efficiently take up GFP
from the body cavity and accumulate GFP in large vesicles. Fluorescent micrograph of wild-type and typical cup mutant worms expressing GFP are shown. High magnification image of a wild-type coelomocyte is also shown.

CUP-6/MTM-6 and CUP-10/MTM-9 belong to a family of lipid phosphatases called
myotubularins (Dang et al., 2004). Members of this protein family are
associated with the human diseases X-linked myotubular myopathy and
Charcot-Marie-Tooth type 4B1 and 4B2. MTM-1 and MTM-3, other worm myotubularin
homologues, are also required for coelomocyte endocytosis (Xue et al., 2003). Several myotubularins have been demonstrated to specifically
dephosphorylate phosphoinositide 3-phosphate [PI(3)P], a key lipid regulator of the
endocytic pathway that is normally enriched in early endosomal membranes (Stenmark and Gillooly, 2001). RNAi of C. elegansVPS-34,
a homologue of the primary PI-3 kinase, also causes a Cup phenotype, suggesting that
the balance of PI(3)P production and degradation is important for regulating
coelomocyte endocytosis (Fares and Greenwald, 2001b). MTM-6 and MTM-9
form a complex, and both mtm-6 and mtm-9
mutants show defects in a very early step of endocytosis. Although both MTM-6 and
MTM-9 possess the lipid phosphatase domain, MTM-9 lacks key residues thought to be
essential for PI-3 phosphatase activity. MTM-9 may play a regulatory role in
controlling the phosphatase activity of MTM-6 (Treusch et al., 2004).

It is still unclear to what extent clathrin-dependent endocytosis and
clathrin-independent pinocytosis mechanisms contribute to uptake of plasma membrane
components and extracellular fluid in coelomocytes. However,
dyn-1 and a set of rme mutants
(rme-1, -4, -6
and -8) show clear defects in coelomocyte endocytosis in
addition to defects in yolk uptake by oocytes (Fares and Greenwald, 2001; Grant et al., 2001; Zhang et al., 2001).
These results indicate that the oocyte and coelomocyte pathways share some
components. However, oocyte-specific rme mutants and coelomocyte specific
cup mutants have been isolated, indicating that not all
components are shared between these cells/pathways (Fares and Greenwald, 2001b; our unpublished observation).

1.3. Signaling and endocytosis

Signaling receptors are thought to be regulated in part through regulated
endocytosis. In some cases endocytosis is thought to down-regulate signaling, while
in others endocytosis may be required for signaling receptors to contact their
downstream effectors located on endosomal membranes. The LIN-12 receptor is
primarily associated with the apical membranes of the VPC cells where it specifies
the secondary cell fate (see RTKRas/MAP kinase signaling). The signaling activity of LIN-12 has been
shown to be developmentally down-regulated in P6.p by endocytosis (Shaye and Greenwald, 2002). LIN-12 endocytosis is triggered in P6.p by a signal
(LIN-3) from the anchor cell received by LET-23 and transduced by Ras (see RTKRas/MAP kinase signaling). Furthermore signaling by the LET-23 EGF
receptor is downregulated in P5.p and P7.p in response to the LIN-12 mediated
lateral signal, probably in part by increased expression of LET-23 associated
endocytosis factors (Yoo et al., 2004).

2. The secretory pathway

Most work on the mechanisms of secretion in C. elegans has
focused on neuronal cells, and more specifically on the mechanism of synaptic vesicle
exocytosis, and to a lesser extent synaptic vesicle biogenesis and axonal transport
(see Synaptic function). Much less is known about general or regulated secretion in other
C. elegans cell types (Figure 5).

Figure 5. General model of vesicular transport pathways. Transport between organelles is mediated by vesicles. Vesicle formation at various steps is driven by specific coat protein
complexes. The C. elegans genome contains genes encoding all of these coat proteins, although some of them have not been studied functionally.

As in other eukaryotes C. elegans cells possess an endoplasmic
reticulum into which most membrane or secretory proteins are inserted
co-translationally. Some lumenal ER-resident proteins are retained in the ER by KDEL or
HDEL signals in their extreme C-termini. C. elegans cells also
possess Golgi stacks, the next organelle through which secretory proteins pass. Unlike
mammalian cells, invertebrates such as C. elegans have many small
"mini stacks" throughout
the cytoplasm of most cells rather than one large stack positioned near the nucleus.
From the Golgi, secretory proteins and some membrane proteins proceed to either the
plasma membrane or to the endosomal system, depending upon various signals embedded in
the primary sequence of the proteins. C. elegans does not possess
any obvious homologs of the mannose-6-phosphate receptors, and so may not use the
mannose-6-phosphate system for tagging and sorting newly synthesized lysosomal
hydrolases. Worms may instead use amino acid based signals and a vps10/sortillin
receptor type system for such sorting, as is known to be the case in yeast.

3. General secretion

Genome-wide RNAi experiments indicate that loss of general secretory factors often
results in embryonic lethality and/or larval lethality associated with developmental
arrest during molting (Grant and Hirsh, 1999). Analysis in the germline
indicates that general secretory defects also result in abnormal oogenesis and often
result in multinucleate oocytes, presumably reflecting a defect in cytokinesis (Grant and Hirsh, 1999; Roberts et al., 2003). In addition
oocytes lacking normal secretory function can produce embryos lacking a strong eggshell
or lacking an eggshell altogether (Grant and Hirsh, 1999). This was not
unexpected as the eggshell is thought to form by secretion of eggshell components by the
embryo itself shortly after fertilization.

Several other proteins associated with cytokinesis are known or assumed to function in
membrane traffic. These include the endocytic protein dynamin and the syntaxin (t-SNARE)
homolog SYN-4 (Jantsch-Plunger and Glotzer, 1999; Thompson et al., 2002). Both of these proteins are associated with cleavage furrows in dividing
C. elegans germ cells and embryonic cells and are required for
completion of cytokinesis (see Cell division). Many proteins associated with the midbody
during cytokinesis are thought to have functions in membrane trafficking (Skop et al., 2004).

One component of the general secretory pathway that has been studied in significant
detail in C. elegans is SEC-23, a part of the endoplasmic reticulum
vesicle coat complex known as COPII (Figure 5). COPII is known to be the primary vesicle
coat complex used in yeast and mammalian cell transport from the ER to the Golgi.
Roberts et al. identified a single mutant allele of sec-23 in a
screen for embryonic lethals defective in cuticle synthesis as assayed by a cuticle
collagen reporter, col-12::GFP (Roberts et al., 2003). These authors went on to show severe defects in cuticle synthesis in
sec-23 mutants resulting in accumulation of DPY-7 collagen
intracellularly, presumably in the ER. Zygotic embryonic lethality during elongation was
found in sec-23 homozygous mutants derived from heterozygous
mothers. Normal progression through early development presumably relies upon maternally
derived SEC-23. RNAi experiments revealed a requirement for sec-23
during larval development, particularly during molting. Adults depleted of SEC-23 by
RNAi also showed severe germline defects including binucleate oocytes, lack of yolk
uptake by oocytes resulting from a failure of yolk receptors (RME-2) to reach the cell
surface, and premature maturation/partitioning of individual germ cells, possibly
resulting from loss of cell surface GLP-1 receptors in the distal gonad. A partially
functional SEC-23::GFP reporter gene indicated that SEC-23 is broadly expressed at all
life stages, and that in hypodermal cells the protein is concentrated in distinct foci.
In the embryonic hypodermis these foci were enriched apically at the periphery of the
endoplasmic reticulum. These SEC-23 positive foci likely represent ER exit sites where
newly synthesized cargo molecules concentrate and are packaged into COPII coated
vesicles for delivery to the Golgi.

Another class of C. elegans proteins thought to function in
secretion are those related to the putative hedgehog receptor Patched. Kuwabara and
colleagues have identified 29 patched (PTC) or patched related (PTR) genes in the
C. elegans genome, each of which encodes a predicted multipass
transmembrane protein with a predicted sterol-sensing domain (Kuwabara et al., 2000). The phenotypes of ptc-1 mutants indicate a likely
defect in the secretory pathway and in germline cytokinesis in particular. The presence
of such a large and diverse family of these proteins in C. elegans
could indicate diverse functions in membrane trafficking processes (Kuwabara et al., 2000).

4. Polarized secretion

Polarized cells such as epithelia maintain distinct apical and basolateral plasma
membrane domains with distinct protein and lipid compositions. These specializations of
the plasma membrane require complex membrane trafficking pathways thought to include
unique basolateral versus apical sorting mechanisms in the secretory pathway to direct
newly synthesized proteins to the correct membrane domain. Additional regulation of the
endocytosis pathway maintains polarity of lipid and protein components after
internalization and recycling.

Ectodermal cells with epithelial characteristics are an important class of polarized
cells that have come under intense scrutiny in C. elegans in recent
years. These include epidermal cells (hypodermal cells), vulval and rectal epithelia,
glia-like sheath and socket cells that support sensory neurons of the amphid and phasmid
sensory organs, and the excretory cell, a single cell that forms a renal-type organ,
extending a long cytoplasmic process called the excretory canal on each side of the
animal.

4.1. Apical secretion

A large number of mutants (exc genes) that display defects in
apical membrane formation in excretory cells have been identified (Buechner et al., 1999). Some of these are likely to function directly in membrane
transport to form or maintain apical membranes. A likely candidate for mediating
vesicular transport is EXC-4, a member of the CLIC chloride channel family (Berry et al., 2003). EXC-4 localizes to vesicles that fuse together to form
the apical membrane of the lumen in the excretory canals. Furthermore
exc-4 is continuously required for tube formation and
maintenance. Although the precise mechanisms remain unclear, EXC-4 may function in
apical secretory vesicle fusion to promote tube formation.

All of the ectodermal epithelial-like cells are known to require the
lin-26 transcription factor to aquire their final
differentiated forms (Labouesse et al., 1994). In particular it is the
apical membrane domain that appear most severely affected in
lin-26 mutant ectodermal epithelia. LIN-26 has therefore been
proposed to regulate the expression of gene products that form and maintain apical
epithelial character. Screens for mutants with Lin-26-like defects could therefore
identify apical-specific trafficking factors. Michaux et al (Michaux et al., 2000) took just this approach and identified CHE-14, a predicted 12-pass
transmembrane protein required for the function of these cell-types.
che-14 mutants displayed defects in the hypodermis,
excretory canal, vulva, rectum and amphids and phasmids. In
che-14 mutants vesicles and amorphous material accumulate near
the apical surface of the affected cells suggesting that secretion is defective.
CHE-14 contains a sterol-sensing domain, a type of domain found in proteins such as
Dispatched, Patched and PC1 that are involved in cholesterol associated trafficking
processes. A rescuing CHE-14::GFP reporter is localized to the apical surface of
epithelial cells that require che-14 function, consistent with
such a proposed function (Michaux et al., 2000).

4.2. Basolateral secretion

Basolateral-specific transport pathways are also thought to play key roles in
polarized cells. The best studied of these pathways in C.
elegans is in the transport of the LET-23 EGF-receptor in the vulval
precursor cells (VPCs; see RTKRas/MAP kinase signaling). LET-23 is normally restricted to the
basolateral membrane of the VPCs, a process that requires the PDZ-domain proteins
LIN-2, LIN-7, and LIN-10 (Kaech et al., 1998). The LIN-2/7/10 complex
binds directly to the LET-23 intracellular domain, and is therefore likely to
function as a sorting factor, directing LET-23 to the basolateral membrane during
secretion and/or after endocytosis and recycling (Kaech et al., 1998).
The LIN-10 protein has been shown to associate with an intracellular compartment on
or near the Golgi consistent with a role in protein sorting during secretion (Whitfield et al., 1999).